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hierarchies among pseudo-clonal honeybee workers (Apis
mellifera capensis)
Stephan Härtel, Theresa Wossler, Gert-Jan Moltzer, Robin Crewe, Robin
Moritz, Peter Neumann
To cite this version:
Pheromone-mediated reproductive dominance hierarchies
among pseudo-clonal honeybee workers
(
Apis mellifera capensis)
Stephan HÄRTEL1,Theresa C. WOSSLER2,3,Gert-Jan MOLTZER4,Robin M. CREWE3, Robin F. A. MORITZ3,4,Peter NEUMANN5,6
1
Department of Animal Ecology and Tropical Biology, Theodor-Boveri-Institute, Biocentre, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
2Centre for Invasion Biology, Department of Botany and Zoology, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa
3Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, South Africa 4Institut für Biologie, Molekulare Ökologie, Martin-Luther-Universität Halle-Wittenberg, Hoher Weg 4, 06099 Halle
(Saale), Germany
5Swiss Bee Research Centre, Agroscope Liebefeld-Posieux Research Station ALP, 3033 Bern, Switzerland 6Department of Zoology and Entomology, Rhodes University, Grahamstown 6140, South Africa
Received 23 June 2010– Revised 16 December 2010 – Accepted 4 February 2011
Abstract– Honey bee colonies are characterised by well-developed reproductive division of labour between the queen and workers. Here, we test whether this reproductive division of labour is evident in both the socially parasitic workers that invade a colony as well as in their offspring generation. We infected six Apis mellifera scutellata host colonies with pseudo-clonal socially parasitic Cape honeybee workers (Apis mellifera capensis). We show that the first generation of socially parasitic workers can monopolize reproduction within host colonies. Of the initially invading parasites, 94.4% became reproductive pseudoqueens with activated ovaries and produced queen-like pheromones. Their offspring, however, had much lower levels of ovary activation (3.1%), yet 89% showed fatty acid synthesis typical of the queen substance (9-oxo-2(E)-decenoic acid) biochemical pathway. However, in these second-generation workers, the last oxidation step from the precursor (9-hydroxy-2(E)-decenoic acid) to the queen substance was interrupted and appears to be required for reproductive dominance in honeybee workers. Our data show that despite the absence of genetic diversity, residual queen mandibular pheromone (QMP) variation is sufficient to establish reproductive dominance hierarchies among parasitic workers. Consequently, QMP produced by a group of workers can maintain reproductive division of labour in queenless honeybee colonies.
Cape honeybees / self-organisation / queen mandibular pheromone / response thresholds / division of labour / worker reproduction
1. INTRODUCTION
Eusocial insect colonies are characterised by reproductive division of labour between the queen and the workers (Wilson1971). In the honeybee,
Apis mellifera, regulation of reproduction is well understood and the reproductive dominance of the queen is mediated by chemical communica-tion via pheromones. Worker reproduccommunica-tion is rare under queenright conditions and the occasional eggs produced by workers are swiftly removed by worker policing (Ratnieks and Visscher
1989). However, whenever a colony cannot replace a lost queen (hopelessly queenless
condi-Corresponding author: S. Härtel, stephan.haertel@uni-wuerzburg.de Manuscript editor: Bernd Grünewald
Apidologie (2011) 42:659–668
Original article
tion), worker policing is curtailed and some workers develop into pseudoqueens, by activating their ovaries, secreting queen-like pheromones and eventually producing their own offspring (Crewe and Velthuis 1980; Ruttner and Hesse
1981; Hepburn 1992; Wossler 2002; Velthuis et al. 1990). The chances of becoming a pseudo-queen are nevertheless not equal among workers. The queen mates with 10–20 drones resulting in multiple worker subfamilies in the colony (Estoup et al. 1994). Members of some sub-families have a higher probability than others of developing as pseudoqueens (Moritz et al.1996; Martin et al.2004; Härtel et al.2006a; Makert et al. 2006), or queens (Moritz et al. 2005b), suggesting high genetic variance for reproductive dominance among worker subfamilies. Aworker’s position in the reproductive dominance hierarchy is largely mediated by her mandibular secretions, and the more queen-like the secretions, the higher is the worker’s position (Moritz et al.2000,2004; Simon et al.2005; Dietemann et al.2006,2007). Moreover, it has been shown that the expression of queen-like mandibular pheromones by work-ers has a strong genetic component (Moritz and Hillesheim1985; Lattorff et al.2005,2007).
The fatty acids of the queen mandibular gland pheromone (QMP) cause changes in both behav-iour (releaser function) and physiology (primer function) of workers (Slessor et al.1988; Winston et al. 1989; Naumann et al.1991; Winston and Slessor1992; Keeling et al.2003; Hoover et al.
2003; Slessor et al.2005). The major component of QMP is the so-called “queen substance” (9-keto-2-(E)-decenoic acid, 9-ODA) (Barbier and Lederer 1960; Butler et al. 1961; Pain
1961) whereas 10-hydroxy-2-(E)-decenoic acid (10-HDA) and 10-HDAA (10-hydroxydecanoic acid) are the major components of the worker mandibular pheromone. The two caste specific biochemical pathways of the mandibular gland pheromones are derived from the same ω-hydroxy-18-carbon fatty acids by chain reduction towards ten carbons (Plettner et al.1996,1998).
Pseudoqueens live for 3 to 5 months (Velthuis et al.1990) approximately three times longer than non-reproductive workers. Pseudoqueens produce queen-like mandibular gland pheromones with
9-ODA dominating the secretion (Hemmling et al.
1979; Crewe and Velthuis 1980; Plettner et al.
1993). A pseudoqueen will prevent other workers from becoming pseudoqueens with its queen-like pheromonal signal, but it has attained pseudo-queen status, it is not affected by the pseudo-queen-like signals of others (Simon et al.2005; Hoover et al.
2003; Dietemann et al.2007).
QMP-meditated dominance hierarchies between pseudo-clonal A. mellifera capensis workers and their offspring during the course of invad-ing A. mellifera scutellata host colonies. Al-though the effect of QMP on worker reproduction has widely been studied, data on the pheromonal regulation of worker exocrine secretions are scanty (Katzav-Gozansky et al.
2006). In this study, we use the parasitic pseudo-clone worker lineage to investigate the development of pseudoqueens and dominance hierarchies in genetically uniform groups.
2. MATERIALS AND METHODS 2.1. Sampling of the experimental worker
groups
Unrelated queenright test colonies of A. mellifera scutellata (n=6) were obtained from their native range (Ixopo, KwaZulu Natal, South Africa, Hepburn and Radloff1998) and placed 1 m apart in a test apiary in Grahamstown (Eastern Cape). Offspring of the ubiq-uitous socially parasitic A. mellifera capensis pseudo-clone (n=10 colonies) (Baudry et al.2004; Härtel et al.
2006b; Neumann et al.2011) were obtained from A.
mellifera scutellata host colonies (n=6) which were in late stages of infestation (reviewed by Neumann and Hepburn 2002; Härtel et al. 2006b) from Pretoria (Gauteng). The test colonies were fed ad libidum with a mixture of honey, pollen and sugar water.
2.2. Experimental set-up
Sealed A. mellifera capensis parasitic worker brood and the A. mellifera scutellata worker brood frames were incubated at 35°C until adult emergence. The emerging A. mellifera capensis parasitic workers (N=700) and A. mellifera scutellata (N=300) were individually marked and introduced into the six test A. mellifera scutellata colonies. In order to secure a high rate of acceptance, we sprayed the test workers with a honey sugar/water solution just before introducing them. All host colonies were successfully infested by the social parasites. One week after the introduction, 150 of the 700 introduced A. mellifera capensis parasites were still present in the host colonies. Over
a period of 6 weeks, the colonies were monitored for the number of remaining labelled test workers. Parasitic worker samples were taken weekly with 12±7 workers collected. The A. mellifera scutellata host workers were sampled at 2 (N=11 workers) and 4 weeks (N=11 workers) after their introduction.
After 4 weeks, brood frames containing offspring of the introduced parasitic workers were removed from the now queenless A. mellifera scutellata test colonies (n= 3; Härtel et al. 2006b) and incubated until adult emergence. The newly emerged offspring were marked and reintroduced into their mother colonies. Again the offspring workers were collected on a weekly basis for up to 6 weeks. In both generations of parasites (initially introduced workers N=35, offspring N=11), their possession of the ubiquitous parasitic worker genotype was confirmed via DNA microsatellite genotyping (Baudry et al. 2004; Härtel et al.2006b, Neumann et al. 2011) using six polymorphic loci (A14, A28, A35, A88, A113 and B124).
2.3. Ovarian activation
In order to analyse the reproductive status of the sampled workers, the abdomens were dissected and the developmental stage of the ovaries was assessed using standard criteria (Velthuis 1970): I=no activa-tion, II=round or bean-shaped eggs visible (early stage of activation) and III=fully developed ovarioles with mature eggs.
2.4. Mandibular gland pheromone development
The sampled workers were decapitated and their heads were extracted in 200 μL dichloromethane (DCM) for spectroscopy (Merck, Uvasol). The extracts were evaporated just to dryness under a stream of N2 and the acids in the extracts were
column (25 m × 0.32 mm × 0.52 μm) which was temperature programmed at 60°C (1 min), 50°C/min to 100°C and 3°C/min to 220°C (10 min). The injection port was set at 230°C while the detector temperature was 280°C. The mandibular gland com-pounds were identified by comparing their mass spectra with those of pure compounds accessed via the Wiley database or by comparisons of the retention times with authentic standard compounds using HP Chemstation software. The amounts of the individual produced mandibular gland components (ODA, 9-HDA, 10-HDA and 10-HDAA) were quantified per sampled worker. A standard solution containing the 9-ODA and 10-hydroxy-(E)-decanoic acid (10-HDA) was run daily to ensure that relative mass ratios were within the limit of variability found in the series of standard runs (Crewe and Moritz 1989). We deter-mined the bias towards the queen substance or worker substance pathway by measuring the ratio of queen-like substances (9-ODA and 9-HDA) and worker-like substances (10-HDA and 10-HDAA) in relation to the total analysed mandibular gland components; total mandibular components TMC= (9-ODA+9-HDA+10-HDA+10-HDAA). The phero-mone ratios data were arcsine transformed and analysed with Statistica©.
3. RESULTS
We did not find a significant difference in the age of the sampled socially parasitic parental and offspring workers (parental, 25.8 ±11.2 days, n=72; offspring, 28.2±10.2 days, n=32).
3.1. DNA genotyping
The DNA microsatellite analysis revealed that of all tested A. mellifera capensis workers (both the initially introduced workers (n=35) and their first offspring (n=11)) belonged to the ubiquitous distributed pseudo-clonal genotype (Baudry et al. 2004; Härtel et al. 2006b; Neumann et al.2011).
3.2. Pheromonal development
The 9-ODA production of the first generation of introduced A. mellifera capensis workers (n= 72; 85.5±15.4% SD) was significantly higher (Mann–Whitney U test, U=68.5 and P<0.05) than that of their offspring (n=32; 29.1±21.3%; Figure 1). The parasitic offspring produced more 9-HDA (60.0±22.4%; U=139 and P< 0.05) than the parental parasites (13.6±1.6%; Figure1). The parasitic offspring (89.1±23.6%) produce significantly less queen-like mandibu-lar substances (9-ODA +9-HDA; Figure 1; U= 691 and P>0.001) than the parental generation (97.6±8.2%).
The parasitic offspring however, produced significantly more worker-like substances in their mandibular glands compared with the initially introduced parasitic workers (10.9± 23.9% and 2.4±8.2%; U=691 and P<0.001). In contrast, the A. mellifera scutellata host workers produced only traces of 9-ODA (n= 18; 6.1% ± 20.7). The worker-like alipathic mandibular gland acids (10-HDA+10-HDAA/
9-ODA 9-HDA
Parental Offspring Host
0 20 40 60 80 100 TMC [ %]
9-ODA+9-HDA +10-HDA+10-HDAA) domi-nated the mandibular gland secretions of A. mellifera scutellata workers 73.9±21.8%.
3.3. Ovarian activation
The first generation of parasitic A. mellifera capensis workers (N=72) showed significantly more activated ovaries compared with their offspring and the A. mellifera scutellata host workers (Kruskal–Wallis ANOVA, df=2, H= 98.49 and P<0.001). While 94.4% of the initially introduced parasites had fully activated ovaries (stage III), only a single worker of the parasitic offspring had fully activated ovaries. All dissected A. mellifera scutellata host workers (N =22) were classified as ovarial stage I (Figure2).
3.4. Correlation between 9-ODA and ovarian activation
The proportion of 9-ODA in the total pheromone blend (9-ODA/9-ODA + 9-HDA + 10-HDA+10-HDAA) showed a significant corre-lation with the ovarian activation in A. mellifera capensis parasitic workers (n=104, R=0.799 and P<0.01; Figure 3). There was no significant difference in the proportion of 9-ODA present in the pheromone blend of individuals with stage I
and II ovarian activation (n=35 and P>0.05; Figure3).
4. DISCUSSION
The first generation of introduced parasitic workers monopolized reproduction in the in-vaded colonies. However, only 3.1% of the parasitic offspring workers, with identical gen-otypes to those of the introduced parasitic workers, and none of the A. mellifera scutellata host workers developed into reproductive pseu-doqueens. The extreme differences in the phenotypes of pseudo-clone workers show that genetic variance is not essential to initiate the self-regulatory process of reproduction and sterility among workers. Our study also shows that the variation in mandibular gland phero-mone concentration is sufficient to regulate the process of separating reproductive from subor-dinate workers. This regulatory mechanism contributes to the residual colony fitness after queen loss due to the establishment of repro-ductive division of labour between pseudo-queens and their offspring workers.
The first generation of A. mellifera capensis workers activated their ovaries in the presence of a laying A. mellifera scutellata host queen, while the host workers did not. Obviously, the response threshold to the queen’s pheromones is
100
75
50
25
0
I II III I II III I II III
I II III I II III I II III
Ov aries [%] Parental ovarial stage Offspring ovarial stage Host ovarial stage
Figure 2. The stages of ovarian activation (means±SE) of the parental generation of socially parasitic workers (n=72 and n=6 A. mellifera scutellata host colonies), their pseudo-clonal offspring (n=32 and n=3 host colonies) and A. mellifera scutellata host workers (n=22 and n=3 host colonies) are shown.
different in the parasite and host worker groups with the parasitic workers not being affected by the QMP of the host queen. Variance for suppression thresholds of individual workers has been repeatedly reported (Oldroyd et al.
1994; Pankiw et al. 2000; Hoover et al. 2005; Dor et al.2005, Dietemann et al.2006). In this study, the higher threshold for the queen pheromone in A. mellifera capensis workers compared with the A. mellifera scutellata work-ers indicate genetic differences between the two subspecies for this trait. This difference is fundamental and predisposes Cape honeybee workers to act as social parasites in A. mellifera scutellata colonies with well known devastating consequences for beekeeping (Allsopp and Crewe 1993; Greeff 1997; Neumann and Hepburn 2002; Moritz et al. 2005a; Dietemann et al.2006). The queen-like pheromone secretion of the first generation of A. mellifera capensis social parasites (Figure1) is consistent with the laboratory results obtained by Dietemann et al. (2006) who showed that pseudo-clone workers produced queen-like mandibular gland secretions even in the presence of A. mellifera scutellata
mandibular queen signals. In addition, also under field conditions our data clearly show that the ovary activation in pseudo-clone workers is not suppressed by the host queen.
The very different fate of first generation social parasites (developing into pseudoqueens) and their offspring (remaining sterile) provides further insight into the self-regulatory process of queen pheromone development through work-er–worker interactions (Moritz and Crewe
2005). Since QMP inhibits ovary activation and QMP production in other workers (Moritz et al. 2000; Dietemann et al. 2007), these hierarchies are most likely established due to changing queen pheromone levels in the colony during the course of invasion. In the initial phase, the pheromone signal of the resident A. mellifera scutellata queens is not sufficient to regulate the reproductive development of intro-duced parasitic A. mellifera capensis workers. Consequently, parasitic pseudoqueens can de-velop rapidly after invasion. When established parasitic pseudoqueens interact with the host queen through lethal fights (Moritz et al. 2003) or through pheromonal competition with the resident queen (Dietemann et al.2006), they are capable of taking over their host colonies. Our data explain observations of previous studies in that only a small number of socially parasitic workers reach reproductive development within infected host colonies (Martin et al.2002; Swart
2003). We show that small groups of invading parasitic workers or the first offspring of a single invading worker can monopolize worker repro-duction in the host colony, thereby establishing reproductive dominance hierarchies among pseudo-clone honeybee workers (Figures 1, 2
and 3).
Although reproduction is suppressed in the parasitic offspring, they rarely take part in worker tasks such as foraging (Martin et al.2002). Since genotypic variance is an important factor in the maintenance of a honeybee colony (recently reviewed by Oldroyd and Fewell 2007), it is not surprising that even non-reproductive A. mellifera capensis workers of this particular socially parasitic pseudo-clone do not take over all tasks to the same degree as host workers do.
75 75 50 25 0 50 25 0 I 100 100 I IIII IIIIII 9-OD A/ TMC [%] Ovarial Stage
This appears to be one of the main reasons for the eventual death of the host colony subsequent to the initial invasions (Neumann and Hepburn
2002; Härtel et al.2006b).
It could be demonstrated by Lattorff et al. (2007) that queen-like mandibular secretions of honeybee workers are strongly linked to the Th locus. Indeed, about 95% of the pseudo-clonal parasitic workers follow the biochemical path-way towards the queen substance (Figure 1). However, the offspring generation could not finalize the biochemical pathway to the end product 9-ODA, indicating the influence of the established reproductive workers of the parental generation. Non-reproductive workers show only low proportions of 9-ODA in their man-dibular gland secretions while their ovaries are not fully developed (Figure 2), resulting in a strong correlation between 9-ODA production and ovary activation (Figure 3). Our findings therefore point to 9-ODA as a fertility signal in socially parasitic honeybee workers. This fertil-ity signal could open a route to the social pathway of worker reproduction (Schäfer et al.
2006).
Workers that track the biochemical pathway of queen substance production but do not show complete ovary activation are most likely able to infect new host colonies. In these workers, the queen-like pheromone production is arrested at the 9-HDA stage but may be predisposed to reproductive dominance. Traits for reproductive dominance of workers are pleiotropically linked to the thelytoky locus (Lattorff et al. 2007). Individ-uals homozygous for the Th allele show both worker ovary activation and queen-like man-dibular secretions. Under the condition of invading a new host colony a reproductive head start is plausible because these pseudo-clone social parasites that have been selected for reproductive traits (Dietemann et al.2007; Neumann et al. 2011) can easily reach repro-ductive dominance in the new host colony.
Workers producing queen-like mandibular secretions have been shown to suppress mandibular gland signal development in neighbouring workers during laboratory studies
(e.g. Moritz et al. 2000; Dietemann et al.
2007). However, our study is the first to suggest the relevance of this phenomenon in the field. Our data highlight that parasitic honeybee workers under queenless conditions use queen-like mandibular pheromones to establish a higher rank in the reproductive dominance hierarchies of the infected colony. These hierarchies are most likely the result of self-regulatory mechanisms of worker–worker interactions through differential response thresholds to 9-ODA concentrations controlled by at least two feedback loops (Moritz and Crewe 2005). The pseudo-clone parasitic off-spring utilise the biochemical pathway leading to the queen substance but there is a second regulatory point that inhibits the final oxidation step to the actual queen substance 9-ODA. As a consequence, the pseudo-clone worker off-spring remain sterile and reproductive division of labour appears to be an inevitable feature of queenless honeybee workers even in the absence of genetic variance, which normally facilitates this process.
In conclusion, our findings support the significant role of mandibular queen pheromone production in the observed reproductive divi-sion of labour of queenless honeybee colonies. The within-colony dynamics of pheromone levels are likely to be the central proximate mechanism for regulating which individuals will be reproductive, whereas the genetic differ-ences among the workers predispose them to become reproductive or remain sterile. At the ultimate level, it is the local intracolony envi-ronment to which the individual is exposed to that determines whether it will develop into a pseudoqueen or not.
ACKNOWLEDGEMENTS
anonymous referee for helpful comments on an earlier version of the manuscript.
Orientation phéromonale de la hiérarchie de dominance reproductive chez les abeilles ouvrières pseudo-clonales (A. mellifera capensis).
abeille du Cap / phéromone mandibulaire / reine / seuil de réponse / reproduction des ouvrières / division du travail
Pheromon vermittelte reproduktive Dominanz-hierarchien zwischen pseudo-klonalen Arbeiterin-nen der Kaphonigbiene.
Kaphonigbiene / Selbstorganisation / Königinnen Mandibelpheromon / Resonanz-Schwellenwert / Reproduktive Arbeitsteilung / Arbeiterinnen Reproduktion
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